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【摘要】 The vascular actions of endothelin-1 (ET-1) reflect the combination of vasoconstrictor ET A and ET B receptors on smooth muscle cells and vasodilator ET B receptors on endothelial cells. The present study investigated the contribution of ET receptor subtypes using a comprehensive battery of agonists and antagonists infused directly into the renal artery of anesthetized rats to evaluate the actions of each receptor class alone and their interactions. ET-1 (5 pmol) reduced renal blood flow (RBF) 25 ± 1%. ET A antagonist BQ-123 attenuated this response to a 15 ± 1% decrease in RBF ( P < 0.01), indicating net constriction by ET B receptors. Combined receptor blockade (BQ-123+BQ-788) resulted in a renal vasoconstriction of 7 ± 1% ( P = 0.001 vs. BQ-123), supporting a constrictor action of ET B receptors. In marked contrast, the ET B antagonist BQ-788 enhanced the ET-1 RBF response to 60 ± 5% ( P < 0.001), suggesting ET B -mediated net dilation. Consistent with ET A blockade, the ET B agonist sarafotoxin 6C (S6C) produced vasoconstriction, reducing RBF by 23 ± 5%. Dose-response curves for ET-1 and S6C showed similar degrees of constriction between 0.2 and 100 pmol. Both antagonists (BQ-123, BQ-788) were equally effective at threefold lower than the standard doses, suggesting complete inhibition. We conclude that ET B receptors alone exert a net constrictor effect but cause a net dilator influence when costimulated with ET A receptors. Such opposing actions indicate more complex than additive interaction between receptor subtypes. Model analysis suggests ET A -mediated constriction is appreciably greater without than with costimulation of ET B receptors. Possible explanations include ET-1 clearance by ET B receptors and/or a dilator ET B receptor function that counteracts constriction.
【关键词】 vascular smooth muscle cells endothelial cells renal vascular resistance nitric oxide
ENDOTHELIN -1 ( ET -1) PLAYS an important role in the regulation of renal hemodynamics and salt and water excretion ( 38 ). Under physiological conditions the vascular endothelium constitutively produces ET-1. Although endogenous ET-1 normally has a minor influence on renal hemodynamics ( 37 ), it gains more importance in disease states such as hypertension, acute and chronic renal failure, and congestive heart failure ( 38 ). ET-1 is capable of constricting or dilating the renal vasculature depending on the relative distribution and influence of ET A and ET B receptors. In the vascular wall, ET A receptors are limited to smooth muscle cells, whereas ET B receptors are expressed in both endothelial and smooth muscle cells ( 38 ). Both receptor types are coupled to Gq proteins, thereby activating several signaling pathways including phospholipase C and intracellular calcium concentration ( 38 ), which leads to constriction of smooth muscle cells. Activation of endothelial ET B receptors by the same signaling pathways produces vasodilation mediated by nitric oxide and possibly eicosanoids ( 14 ).
There is general agreement that administration of ET-1 produces renal vasoconstriction ( 38 ). However, reports on the relative influence of receptor subtypes are highly variable. This ranges from exclusive mediation by ET A receptors ( 8, 23, 36 ) to only that of ET B receptors ( 12, 25, 30 ), including other studies suggesting a more balanced contribution ( 4, 11, 18, 28, 31, 41 ). Furthermore, there is also controversy about the actions of endothelial ET B receptors, ranging from no dilation ( 10, 12, 18, 27, 40 ) to strong dilator effects of ET B receptors ( 1, 2, 5, 28, 31 ).
The factors governing the relative effects of ET A and ET B receptors and the reasons for the discrepancy of previous findings are difficult to discern. Specific vascular beds, vessel size, species ( 22, 33 ), hydration status ( 34 ), or strength of stimulation ( 34 ) may play a role. Anesthesia does not seem to be a major factor for either the finding of predominant ET A - mediated constriction ( 7, 8, 23, 36 ) or predominant ET B - mediated renal vasoconstriction ( 25, 36 ). However, an important contributing factor seems to be the experimental strategy employed. The contribution of ET A receptors is based mainly on effects of ET A -specific antagonists. The contribution of ET B -mediated constriction is assessed from either the remnant response after ET A receptor inhibition, the effect of ET B receptor inhibition, or direct stimulation with ET B -specific agonists. It appears that the contribution of dilator and constrictor functions of ET B receptors depends on whether ET B receptors are stimulated alone or in conjunction with ET A receptors. Inhibition of ET B receptors usually augments ET-1-induced constriction in rats ( 1, 28, 31 ), rabbits ( 13 ), dogs ( 11 ), and humans ( 5 ), suggesting a net dilator influence of ET B receptors, although exceptions are noted in rats ( 4, 10, 18 ). A primary constrictor action of ET B receptors is suggested by the finding that an ET B agonist reduces renal blood flow (RBF) in rats ( 12, 18, 23, 25, 28, 30, 31, 41 ), mice ( 4 ), and pigs ( 10 ). It is important to appreciate that only few studies have used multiple pharmacological combinations of approaches, including ET A antagonist, ET B antagonist, and ET B agonist in a systematic, comprehensive fashion ( 10, 13, 18, 28, 31 ). However, even those studies do not give a uniform picture. While the apparently opposing influences of ET B receptors were observed in the same preparation in two of those reports ( 28, 31 ), others concluded that the effects of ET B stimulation are limited to either only the dilator ( 13 ) or only the constrictor effects ( 10, 18 ). A limitation of those studies is that drugs were administered intravenously, with the possibility of confounding systemic effects secondarily to changes in arterial pressure, or investigations were performed in the hydronephrotic kidney model ( 18 ) with administration of drugs to the tissue bath, which may differ from the normal situation.
The purpose of our study was to investigate comprehensively the participation of ET receptor subtypes in the renal hemodynamic response to ET-1 by employing a battery of tools (ET A and ET B antagonists, and ET B agonist). Because the tonic influence of endogenous ET-1 is known to be small ( 24 ), we concentrated on the response to exogenous ET-1. To allow a more quantitative analysis of direct renal effects than has been done in previous rat studies, we employed injections of vasoactive agents directly into the renal artery to avoid confounding systemic effects. In addition, we conducted paired studies in that ET-1 responses were evaluated during control, experimental, and recovery periods in the same animal.
METHODS
Experiments were made in 36 male Sprague-Dawley rats (6-8 wk of age, 170-300 g body wt) from our local breeding colony in accordance with institutional guidelines for the care and use of research animals. The animals were fed a standard lab chow with free access to tap water and kept on a 12:12-h light-dark cycle.
Surgical Preparation
After induction of anesthesia by pentobarbital sodium (Nembutal, 50-60 mg/kg body wt ip, Abbott, Chicago, IL), a rat was placed on a temperature-controlled table kept at 37°C. The depth of anesthesia was monitored by the response to ear or toe pinching. The left femoral artery was catheterized (PE-50) for measurement of arterial pressure, and two femoral vein catheters (PE-10) were used for infusion of volume replacement and pentobarbital sodium. The trachea was cannulated (PE-240) to facilitate respiration. Via a midline abdominal incision, the aorta and left renal artery were exposed. A catheter (PE-10 with bent tip) was inserted into the left common iliac artery and advanced until its tip faced the origin to the left renal artery for infusion of pharmacological agents into the renal artery. An ultrasound transit-time flow probe (1RB, Transonic, Ithaca, NY) was placed around the left renal artery and filled with ultrasonic coupling gel (HR Lubricating Jelly, Carter-Wallace, New York, NY, or Surgilube, Fugera, Melville, NY). Urine was drained from the bladder by gravity via a 23-gauge needle. Isoncotic bovine serum albumin (4.75 g/dl) was infused initially at 50 µl/min to replace surgical losses (1.25 ml/100 g body wt), followed by a maintenance rate of 10 µl/min. The renal artery catheter was perfused with normal saline at 5 µl/min. Additional doses of pentobarbital sodium were given (iv) as required. All syringes and catheters in contact with peptides were pretreated with albumin solution (0.5 g/dl) to reduce surface adhesion. At least 60 min were allowed after surgery before the experiments were started.
Measurements
Femoral arterial pressure (AP) was measured via a pressure transducer (Statham P23 DB). Renal blood flow (RBF) was measured by a flowmeter (T 206, low-pass filter, 30 Hz, Transonic). Zero offset was determined at the end of an experiment after cardiac arrest. AP and RBF were recorded on a computer (Pentium III+DataTranslation A/D converter+Labtech Notebook-Pro 10.1) at 100 Hz and stored at 1 Hz as consecutive mean values over 1-s periods. AP was also stored at 100 Hz for later determination of heart rate (HR).
Protocols
The RBF response to a bolus injection of ET-1, sarafotoxin-6C (S6C), or IRL-1620 (IRL) into the renal artery was measured during control conditions as well as during an experimental period, and a recovery period. In the first series of studies, different inhibitors of endothelin receptor subtypes were infused in the experimental period. Five minutes before the bolus injection, the renal arterial infusion rate was increased from 5 to 140 µl/min. A 10-µl bolus of ET-1, S6C, or IRL was then injected into the infusion line by a microinjector valve (Cheminert, Valco Instruments, Houston, TX), and a new recording was started. Because it took 22-24 s for the bolus to travel from the injector to the kidney, the initial 20 s of the recording served as the baseline values of AP and RBF. Ten minutes after the ET-1 bolus, the infusion rate was returned to 5 µl/min. The recording was continued until 30 (ET-1) or 15 min (S6C and IRL) after the injection. To achieve ET receptor inhibition, saline was replaced by an infusion of an antagonist (7 nmol/min). AP and RBF were recorded from 1 min before infusion of an antagonist and continuing for an additional 5 min. Subsequently, a new recording was started simultaneously with intrarenal injection of ET or S6C. Thirty to thirty-five minutes were allowed for recovery after each injection of ET-1 and 15-20 min after S6C or IRL.
Renal hemodynamic effects of ET-1 during inhibition of ET A receptors. To delineate the contribution of ET A receptors, responses to injection of ET-1 (10 µl x 0.5 µM) into the renal artery were recorded during infusion of saline (control), during infusion of ET A receptor inhibition by BQ-123 (7 nmol/min), and again during infusion of saline (recovery) in intervals of 30-35 min, each.
RBF effects of ET-1 during inhibition of ET B receptors. The influence of ET B receptors was tested using the same protocol, except that the ET B -receptor antagonist BQ-788 (7 nmol/min) was used in the experimental period.
RBF effects of ET-1 during combined inhibition of ET A and ET B receptors. To determine the completeness of receptor subtype inhibition, the ET-1 response was assessed during combined infusion of both antagonists (7 nmol/min, each).
Renal hemodynamic response to activation of ET B receptors. ET B receptors were stimulated by injection of the selective ET B -receptor agonist S6C (10 µl x 0.5 µM) or IRL-1620 (10 µl x 0.5 µM).
Time control experiments. To establish similar RBF responses to repeated injections of ET-1, ET-1 was injected three times in intervals of 30-35 min.
Completeness of inhibition by ET A - or ET B -receptor antagonists. Vasoconstrictor responses to injection of ET-1 into the renal artery were blocked by BQ-123 at the standard dose (7 nmol/min) and also at 3 x and at 10 x lower doses (2.3 and 0.7 nmol/min) in random order. Reversibility was confirmed in a recovery period. In other animals, BQ-788 was infused at 7 nmol/min and also at lower doses (2.3 and 0.7 nmol/min) in random order, followed by a recovery period.
Renal vascular effects of S6C before and during inhibition of ET A and ET B receptors. To test the specificity of BQ-123 and BQ-788, reactivity to S6C was evaluated after a control period by infusing either BQ-123 or BQ-788 (7 nmol/min, each), followed by a recovery period.
Dose-response curves for ET-1 and S6C. To test whether dilator effects of ET-1 or S6C might prevail at low concentrations and to compare the responses to selective ET B receptor stimulation to those to stimulation of both ET receptors, each agent was injected into the renal artery in increasing doses (0.02, 0.1, 0.5, 2, and 10 µM x 10 µl) in intervals of 30-35 min. Because of the long-lasting effect of ET-1, the doses were given in ascending order. Doses of S6C were given in ascending order at intervals of 15-20 min.
Drugs and Chemicals
ET-1, S6C, BQ-123, and BQ-788 were obtained from American Peptide (Vista, CA). BQ-788 was also obtained from Peninsula Labs (San Carlos, CA). IRL-1620 was from California Peptide Research (Napa, CA), and albumin was from Sigma (St. Louis, MO).
Data Analysis
The maximum RBF decrease after each injection was determined off-line by custom-built software (AJ) from the 1-Hz data after smoothing by sliding the average over five values. The change was expressed as percentage of the baseline value. Baseline RBF and AP were determined from the average of the first 20 s of each recording immediately before injection. To obtain mean time courses, the original 1-Hz recordings (without smoothing) from all animals in a group were averaged for each experimental period. HR was determined from the 100-Hz recording of AP off-line. Data are expressed as means ± SE. Statistical significance among groups was tested by ANOVA in conjunction with Holm-Sidak's or Tukey's test for multiple comparisons (SigmaStat 3.00, SPSS, Chicago, IL). In the case of nonnormal distribution, data were transformed by square root before analysis. A paired t -test was used to detect changes within a group. P < 0.05 was considered statistically significant.
RESULTS
The age of animals averaged 6.7 ± 0.1 wk, body weight was 252 ± 6 g, and left kidney weight was 1.39 ± 0.04 g. Hematocrit before surgery was 44 ± 1%, and heart rate at the beginning of an experiment was 314 ± 7 beats/min. Group data for baseline AP and RBF are given in Table 1.
Table 1. Baseline hemodynamic data at the beginning of the experiments
Renal Hemodynamic Effects of Endogenous ET-1 Mediated by ET A and ET B Receptors
Renal vascular responses to administration of receptor antagonists are shown in Fig. 1. Inhibition of ET A receptors by infusion of BQ-123 into the renal artery increased RBF by 9 ± 3%. Intrarenal infusion of the ET B antagonist BQ-788 reduced RBF by 9 ± 2%. Infusion of both antagonists together had no net effect on RBF (3 ± 3%, P 0.2). AP and heart rate were stable during these intrarenal infusions, as was the case in subsequent studies. Collectively, these findings indicate that endogenous ET exerts a tonic renal vasoconstriction via ET A receptors and an offsetting tonic dilator tone via ET B receptors, effects that cancel each other out under basal conditions.
Fig. 1. Effects of inhibition of endothelin (ET) receptor subtypes on baseline renal blood flow (RBF). Change in RBF during ET A receptor inhibition by infusion of BQ-123 (7 nmol/min) into the renal artery ( n = 7), during ET B receptor inhibition by BQ-788 (7 nmol/min, n = 7), or during ET A +ET B receptor inhibition by infusion of BQ-123+BQ-788 together ( n = 6) is shown. RBF is expressed as percent change from immediately before to 5-7 min after the start of infusion. Values are means ± SE. * P < 0.05 vs. control.
Renal Hemodynamic Effects of Exogenous ET-1
Injection of ET-1 into the renal artery caused a 25 ± 2% reduction in RBF (in both Fig. 2, A and B ). No dilatory phase was evident. The ET-1 maximum response was reached between 1 and 2 min after injection. Recovery was slow; RBF was 18 ± 1 and 15 ± 2% below baseline at 15 and 30 min, respectively (pooled controls before ET A or ET B inhibition). As a result, the next injection of ET-1 was made before complete recovery. RBF recovery at 30-35 min was regarded as a reasonable compromise between sufficient recovery and length of an experiment. To demonstrate consistent ET-1 responses during repeated stimulation, time control experiments were conducted. To test reversibility of receptor antagonists, ET-1 was given after antagonist during a recovery period.
Fig. 2. Responses of RBF to stimulation and inhibition of specific endothelin receptors. A : RBF responses to injection of ET-1 (5 pmol) into the renal artery during control conditions ( ), during ET A receptor inhibition by BQ-123 (7 nmol/min,, n = 7), and during ET A +ET B receptor inhibition by intrarenal coinfusion of BQ-123 and BQ-788 (7 nmol/min each, solid line, n = 6). B : RBF changes induced by intrarenal injection of ET-1 during control conditions ( ), during ET B receptor inhibition by BQ-788 (7 nmol/min,, n = 7), and during inhibition of ET A +ET B receptor inhibition by intrarenal coinfusion of BQ-123 and BQ-788 (7 nmol/min each, solid line, n = 6). C : RBF responses to selective ET B receptor stimulation by intrarenal injection of sarafotoxin 6C (S6C; 5 pmol, n = 7). Values are means ± SE expressed as percent change from baseline.
Time Control Series
Repeated injection of ET-1 produced similar degrees of renal vasoconstriction over time: 24 ± 4, 26 ± 1, and 27 ± 3% for injections made at 0 min, 30-35 min, and 60-70 min, respectively, even though there was a progressive reduction of baseline RBF (from 4.2 ± 0.3 to 3.3 ± 0.2 ml·min -1 ·g -1, n = 4).
RBF Responses to ET-1 Mediated by ET A Receptors
The ET A -receptor antagonist BQ-123 reduced the constrictor response to ET-1 from 25 ± 2 to 15 ± 1% ( P < 0.001, Fig. 2 A ). The inhibition was reversible as the constrictor response to ET-1 during the recovery period was even larger than during the control period (35 ± 3%, P < 0.001 vs. BQ-123, P < 0.01 vs. control). During ET A inhibition, ET-1 produced transient renal vasodilation lasting <20 s ( Fig. 2 A, ). Nevertheless, the major sustained 15% vasoconstriction due to ET B receptors was larger than that during inhibition of both ET receptors (7 ± 1%, P < 0.001, Fig. 2 A ). Note that recovery was accelerated such that RBF returned to baseline by 10 min ( Fig. 2 A ).
RBF Vascular Effects of ET-1 Mediated by ET B Receptors
During BQ-788 inhibition of ET B receptors, the constrictor response to ET-1 was enhanced more than twofold (-60 ± 5 vs. -25 ± 2%, Fig. 2 B, P < 0.001). The more pronounced renal vasoconstriction was reversible; the ET-1 response during the recovery period returned to values close to control (-33 ± 4% RBF, P < 0.001 vs. BQ-788). This indicates a net vasodilator role of ET B receptors under these conditions. Despite the overall larger response, the recovery rate was not affected by ET B inhibition; RBF recovered by 29 ± 3 and 43 ± 5% after 15 and 30 min during ET B inhibition compared with control values of 22 ± 8 and 36 ± 11%, respectively (data not shown).
Renal Hemodynamic Response to Activation of ET B Receptors
Administration of the ET B -receptor agonist S6C produced obvious renal vasoconstriction ( Fig. 2 C ). The maximum effect (25 ± 3% decrease in RBF) was similar to that seen in response to ET-1 injection (25 ± 2%). The recovery in the S6C group was faster, reaching 70 ± 11% after 15 min compared with 26 ± 6% for ET-1 ( P = 0.001).
Early Hemodynamic Effects of ET-1
During ET A inhibition, ET-1 produced a transient renal vasodilation reaching 3 ± 1% at 30-32 s after injection ( P < 0.05 vs. baseline, Fig. 3 ). ET-1 alone elicited no obvious dilator response. However, during ET B inhibition, the ET-1-induced constriction started 5-10 s earlier than during control ( Fig. 3 ). ET-1-induced RBF reduction reached 10 ± 2% at 30-32 s during ET B inhibition ( P < 0.001), whereas after ET-1 alone RBF was unchanged at the same time point (0 ± 1%, P 0.6, Fig. 3 ). These data suggest a small initial dilator effect of ET-1 that is mediated by ET B receptors.
Fig. 3. Initial responses of RBF to stimulation and inhibition of specific endothelin receptors. Changes in RBF from baseline after injection of ET-1 (5 pmol) into the renal artery alone (, n = 14), during ET A receptor inhibition by intrarenal infusion of BQ-123 (7 nmol/min,, n = 7), and during ET B receptor inhibition by BQ-788 (7 nmol/min,, n = 7). Also shown is the response to selective ET B receptor stimulation with S6C (5 pmol,, n = 7).
Completeness of Inhibition by ET A - or ET B -Receptor Antagonists
To test the efficiency of receptor inhibition using the standard dose (7 nmol/min), receptor antagonists were also infused at lower doses. BQ-123 exerted the same inhibitory effect at the 3 x lower dose but was less effective at 10 x lower dose ( Fig. 4 A ). BQ-788 displayed a similar degree of inhibition for all doses tested. Thus maximum inhibition was likely achieved at the employed standard doses of both antagonists. The ET B agonist IRL-1620 mimicked the constrictor effects of S6C (41 ± 7 vs. 35 ± 10% after S6C), establishing that the constrictor effect was not agent specific.
Fig. 4. Inhibitory effect of endothelin receptor antagonists at different doses on the response of RBF to ET-1. A : percent reduction of RBF in response to ET-1 (5 pmol) injection into the renal artery during control conditions ( left open bar), during intrarenal infusion of ET A receptor antagonist BQ-123 at 3 doses (ranging from 7 to 0.7 nmol/min; hatched bars), followed by ET-1 injection during a recovery period ( right open bar, n = 3). B : RBF responses to ET-1 during control conditions, during infusion of ET B receptor antagonist BQ-788 at three doses (ranging from 7 to 0.7 nmol/min), followed by an ET-1 response during a recovery period ( n = 3). Values are means ± SE. * P < 0.05 vs. respective initial control.
Renal Vascular Effects of S6C Before and During Inhibition of ET A and ET B Receptors
Inhibition of ET A receptors by BQ-123 did not affect ET B receptor stimulation with S6C (24 ± 5 vs. 23 ± 5%, Fig. 5 ). In contrast, inhibition of ET B receptors by BQ-788 attenuated the response to S6C to 8 ± 2 ( Fig. 5 ). Taken together, these results demonstrate that the inhibitors exerted the expected subtype specificity.
Fig. 5. Effect of endothelin receptor antagonists on the response of RBF to S6C. RBF changes in response to injection of ET B receptor agonist S6C (5 pmol) into the renal artery during control conditions, during ET A receptor inhibition (inhib.; BQ-123; 7 pmol/min), during ET B receptor inhibition (BQ-788; 7 pmol/min), followed by the response to S6C during a recovery period are shown. Values are means ± SE, n = 3. * P < 0.05 vs. initial control.
Dose-Response Curves for ET-1 and S6C
Dose-response curves for ET-1 and S6C revealed renal vasoconstriction at doses above 1 pmol. The maximum constrictor effect of both compounds was similar over all doses ( Fig. 6 A ). There was no difference in sensitivity or reactivity to ET-1 or S6C. However, the recovery rate was faster after S6C ( Fig. 6 B ), a difference that was more pronounced at higher doses.
Fig. 6. Dose-response curves for the response of RBF to ET-1 and S6C. Time dependency of RBF responses to injection of ET-1 (, n = 3) or the ET B -receptor agonist S6C (, n = 5). Values are means ± SE. A : immediate maximum constriction observed within 2-3 min after injection. B : sustained RBF response recorded 15 min after the injection.
DISCUSSION
The present study shows that inhibition of ET A receptors reduces by 50% the acute renal vasoconstrictor response to ET-1 injected directly into the renal artery (from -25 to -15% of baseline RBF). The remaining constriction mediated by ET B receptors was antagonized by an ET B receptor blocker. A similar 25% decrease in RBF was observed when ET B receptors were selectively stimulated by the agonists S6C or IRL-1620. In striking contrast, specific inhibition of ET B receptors markedly enhanced ET-1-induced renal vasoconstriction, with a more than twofold decrease in RBF (from -25 to -60%), indicating a pronounced net dilator influence of ET B receptors under these experimental conditions. Collectively, these findings for ET B receptors appear to be contradictory: constrictor at times and dilatory at others. Such duality suggests receptor interactions such that ET B -mediated actions vary as a function of ET A receptor activity and vice versa. An important aspect of our study was that ET and ET-receptor agonists and antagonists were injected directly into the renal artery to restrict acute responses to local changes in vascular resistance of the rat renal circulation independent of extrarenal factors responsive to changes in arterial pressure.
The effects of endogenous ET, based on receptor antagonism during basal conditions, generally agree with the pattern of receptor blockade observed during administration of exogenous ET-1. The ET A -mediated vasoconstriction and the net dilator actions of ET B receptors averaged 5-10% of basal RBF, consistent with most reports that utilized systemic administration of antagonists (for a review, see Ref. 24 ). Because of these small changes, quantitative analysis was done from the responses to exogenous ET-1. Most previous publications investigating the effects of exogenous ET-1 have assessed the function of a single receptor subtype, only. Few have systematically investigated receptor actions and interactions in a comprehensive manner in the same study ( 10, 13, 18, 28, 31 ). In three of these studies, neither the constrictor ( 13 ) nor the dilator component of ET B receptor activation ( 10, 18 ) was found, in contrast to our findings. The reason for the differences is not clear, but may be related, at least in part, to species differences between rats and pigs ( 10 ) or rabbits ( 13 ). The third study was conducted in a chronic hydronephrotic preparation of the rat renal vasculature, and agents were added to the abluminal bath ( 18 ). The site of application may be important as the proposed clearance function of ET B receptors may depend on a concentration gradient for ET-1 from the lumen to the outer vascular wall ( 20 ). In the two in vivo rat studies ( 28, 31 ), results qualitatively similar to our observations were found. A major difference in experimental design was that ET agonists and antagonists were administered systemically in these earlier studies, and the impact of changes in renal perfusion pressure and other extrarenal factors could not be excluded. By design, our more quantitative analysis of local effects utilized intrarenal administration of vasoactive agents. The present in vivo results highlight the local opposing actions of ET B receptor activation in the renal microcirculation, independent of systemic influences. To date, these provocative findings have received relatively little attention and await a unifying explanation. We document that antagonists to ET A and ET B receptors exert near-complete inhibition at the doses employed. Sufficient subtype specificity was demonstrated by the effects of antagonists on the response to S6C ( Fig. 5 ) and the similarity of actions of S6C and IRL stimulation of ET B receptors.
To better understand the apparent discrepancy in results, especially with regard to the opposing actions of ET B receptors, and to gain insight into receptor actions and interactions, three models are presented in Fig. 7. Predicted single-receptor responses are compared with the observed net reductions in RBF. Model A has strictly additive receptor actions such that 1 ) stimulation of all receptors by ET-1 produces 25% renal vasoconstriction and 2 ) the constrictor response to ET-1 is reduced from 25 to 13% during ET A receptor inhibition ( Fig. 7 A ). Clearly, the two additional sets of observations do not fit with model A : 1 ) the 60% constriction produced by selective stimulation of ET A receptors by ET-1 during ET B inhibition is considerably more than the predicted 13% decrease in RBF and 2 ) the 25% decline in RBF elicited by the ET B -receptor agonist S6C is greater than the predicted 13%. Thus some but not all of our major findings can be accounted for by this simple model. No fixed combination of simply additive contributions can explain all of our observations.
Fig. 7. Schematic diagram of predicted and observed responses to endothelin receptor stimulation according to different models. Arrows in downward direction and positive numbers denote vasoconstriction; arrows in upward direction and negative numbers denote vasodilation. Arrow sizes and values give the relative magnitude of the effect reductions of RBF from baseline. Model A assumes strictly additive contributions of the ET receptor subtypes such that an effect of a given receptor is the same under all conditions. Model B assumes that the constrictor effect of ET A receptors varies as a function of ET B receptor activity. Model C assumes that the dilator effect of endothelial ET B receptors varies as a function of the activity of ET A receptors.
Model B ( Fig. 7 B ) is based on the two original assumptions and the additional qualification that ET A receptor-induced constriction is about five times greater during ET B receptor blockade than it is when both ET receptor classes are stimulated simultaneously. Unexplained is the observation that the constriction produced by ET B receptor stimulation with S6C (-25%) is about twice the constriction produced by ET B receptor activation by ET-1+ET A antagonist (-13%). Model C considers the possibility that the renal vasoconstriction by ET A and smooth muscle ET B receptors is modulated to a variable extent by endothelial ET B receptors such that their dilator influence is roughly proportional to the amount of the contemporaneous net constrictor response ( Fig. 7 C ).
Reasonable biological explanations, which are not mutually exclusive, exist to explain variations in the constrictor effect of ET A receptors or the variable dilator effect of endothelial ET B receptors. For example, enhanced ET A receptor-mediated constriction would be expected during ET B receptor blockade ( model B ) if ET B receptors function to clear local ET-1. ET-1 is rapidly removed from plasma with a half-life of <1 min ( 21, 39 ), an action that may be largely, if not exclusively, due to ET B receptors ( 16, 21 ), although the precise anatomic location of the receptors responsible for clearance is not known. Because receptor binding is extremely strong and nearly irreversible, ET-1 may preferentially bind to endothelial receptors exposed to the highest concentration and closest to the production site ( 20 ). Thus disruption of the postulated ET B receptor clearance function may allow more ET-1 to be available to activate ET A receptors. However, the vast majority of plasma clearance occurs in the lung, whereas the kidneys contribute only 10% ( 42 ), and, of this, 50% seems to be independent of ET receptors ( 21 ). Furthermore, the potential impact of renal ET clearance by ET B receptors on renal vascular control is limited by the fact that most ET receptors, particularly the ET B subtype, are located in the renal medulla ( 8, 25 ), separate from cortical resistance vessels. Therefore, it is uncertain whether immediate renal clearance is of sufficient magnitude to account for the observed strong enhancement of the ET A response localized to the renal microcirculation.
A second possibility is that the dilator effect of endothelial ET B receptors is substantially larger during concurrent ET A receptor stimulation than during stimulation of ET B receptors alone ( model C ). Such a buffering action may primarily oppose vasoconstriction without the need to produce vasodilation on its own. In this regard, the magnitude of the dilator-like effect may be covert, absent when there is little constriction to offset, and varying as a function of the strength of concurrent constriction. Accordingly, in the absence of ET A -mediated constriction, a smaller fraction of the ET B dilator-like action would be evident. The magnitude of the buffering depends on the relative contribution of ET A and ET B receptors to the constrictor effect. Previous studies have shown that renal NO can exert strong buffering of constrictor agents without exhibiting net dilation on its own ( 9 ). It is reasonable to propose that such a mechanism is operative in the present study, with endothelial ET B receptors effectively counteracting ET-1-induced renal vasoconstriction. In addition, differences have been reported between the intracellular signaling pathways of ET A and ET B receptors on smooth muscle cells. It is therefore conceivable that the buffering effect of NO may preferentially affect ET A receptor-mediated constriction ( 3 ), which may also explain variable ET B -mediated dilator effects as a function of concomitant ET A receptor stimulation.
A finding not accommodated in any of the models is that the response to S6C or IRL-1620, selective ET B agonists, was almost the same as that to activation of both ET-1 receptor subtypes (25% reduction in RBF). We observed that the renal vasculature exhibited the same sensitivity and reactivity to selective ET B receptor stimulation and to dual-receptor activation by ET-1. There are mixed results on this point in the literature. Previous rat RBF studies report that the relative strength of the vasoconstrictor response to ET B receptor stimulation varies, being two to three times smaller ( 31 ), equal to ( 12, 41 ), or twofold greater than, that of ET-1 ( 25 ). Others report variability depending on dose ( 30 ). It is not clear why S6C and ET-1 produce a similar degree of renal vasoconstriction and why the amount is greater than the response to ET-1 during ET A inhibition. There is an equal distribution of ET A and ET B receptors reported for preglomerular renal microvessels ( 15, 17; unpublished observations). We found that the RBF response to S6C is not affected by ET A antagonism, so tonic stimulation of ET A receptors seems an unlikely explanation as is nonspecific activation of ET A receptors by S6C. Also possible is preferential activation of ET B receptors on smooth muscle vs. endothelial cells by S6C and IRL-1620. Given that ETB receptors are formed from the same gene ( 32 ), differential activation would be surprising. Although indications for slight differences in the susceptibility of the two ET B receptor locations to different ET B antagonists have been reported ( 26, 28 ), the similarity of the responses to ET B agonists S6C and IRL-1620 in the present study speaks against a major difference in sensitivity of endothelial and smooth muscle ET B receptors, at least at the employed doses of ET B agonists. Finally, it has been suggested that the relative influence of the dilator effect of endothelial ETB receptors is smaller or absent at lower doses of ET-1 ( 35, 36 ), possibly due to a higher affinity of endothelial than smooth muscle ET B receptors for ET-1. However, binding curves for ET-1 are typically monophasic, including renal microvascular tissue ( 15, 17 ), thus giving no indication for different affinities of endothelial and smooth muscle receptors. In addition, the dose-response relationships in the present data showed similar strength of ET-1 and S6C responses at all doses and failed to detect a significant dilator effect at any dose ( Fig. 6 ). Accordingly, at least a major shift in the influence of the dilator effect of endothelial ET B receptors with the dose of ET-1 seems unlikely from the present data.
Another noteworthy finding is the faster recovery after stimulation of ET B receptors (ET-1+ET A inhibition or S6C or IRL-1620) compared with stimulation of both ET receptors (ET-1 alone) or stimulation of ET A receptors (ET-1+ET B inhibition). This time course has been reported previously ( 1, 5, 19, 31 ). The reason for the difference in the recovery rate is not clear. In view of the exceptionally tight ligand-receptor binding ( 20 ), the sluggish reversibility may be related to receptor trafficking ( 29 ). Internalized ET A and ET B receptors may experience different fates such as preferential lysosomal destruction of ET B receptors and recycling of ET A receptors to the cell surface ( 6 ).
In conclusion, our comprehensive study provides new insight into the mechanisms by which ET A and ET B receptors contribute to renal vasomotor responses in the normal kidney of euvolemic rats. Our studies highlight a complex interaction between ET A and ET B receptors that impact on their respective function. Selective activation of ET B receptors mediates net renal vasoconstriction. In marked contrast, ET B receptors provide a strong dilator-like influence to buffer constriction produced by ET A receptor stimulation. Opposition of ET A -mediated constriction may be due to the ability of ET B receptors to clear ET from the plasma and lower the effective concentration available for ET A receptors. Another possibility that is not mutually exclusive is that endothelial ET B receptors, presumably via release of endothelial nitric oxide, effectively attenuate the constrictor responses to ET A receptor stimulation rather than eliciting frank dilation on their own. The extent to which ET B receptors on endothelial cells and smooth muscle cells contribute to the proposed interaction by either or both of these mechanisms awaits further investigation.
ACKNOWLEDGMENTS
GRANTS
This work was supported by National Heart, Blood, and Lung Institute Grant HL-02334.
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作者单位：Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545